This document discusses aflatoxins, including their origin, biosynthesis, association with cancer, and regulations. It begins with an introduction to aflatoxins, describing them as secondary metabolites produced by certain fungi. Their classification and health impacts are also covered. The document then discusses the historical background of aflatoxins, their potential fungal sources, and the chemistry and biosynthetic pathway involved in their production. It also reviews factors that can influence aflatoxin synthesis. Next, the association between aflatoxin exposure and liver cancer is examined. Finally, regulatory strategies and techniques for determining aflatoxin levels established by various organizations are summarized.

1. AFLATOXINS: Their Origin, Biosynthesis, Association with
Cancer and Regulations
January, 2018Tesfay H.
Mekelle University
College of Health Sciences
School of Pharmacy
Department of Pharmacognosy

2. Objective
General objective
 To synthesize the current information on aflatoxins (AFTs), their origin,
biosynthesis, association with cancer and regulations
Specific objectives
 To review information provided by relevant articles about
Aflatoxins and their origin
Chemistry and biosynthesis
Association of aflatoxins with cancer
Different regulatory strategies of different organizations and countries and aflatoxin
determination techniques
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3. Methodology
 Electronic databases used
PubMed
Science Direct
Research Gate
Google Scholar
 Searches were limited to articles written in English published by peer reviewed
journals and international organization.
 The search results were assessed for relevance by comparing the tittle, abstract
and the full text.
3
Commonly used search terms
• aflatoxins, mycotoxins, health impacts of aflatoxins
• history of aflatoxins, sources of aflatoxins
• chemistry of aflatoxins, biosynthesis of aflatoxins
• aflatoxins and cancer, association of aflatoxins and cancer,
• determination of aflatoxins, and regulation of aflatoxin
contamination
• Aflatoxin prevalence in Ethiopia, Regulation of aflatoxins in
Ethiopia

4. Outline
 Background, classification and health consequences of aflatoxins
 Origin of aflatoxins (historical background and potential sources)
 Chemistry and biosynthesis of aflatoxins
 Aflatoxins as liver carcinogens
 Regulations of aflatoxins
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5. INTRODUCTION
• What are Aflatoxins?
• Classification of Aflatoxins
• Health Consequences of Aflatoxins
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6. Aflatoxins?
What are aflatoxins?
 Group of secondary metabolites derived from polyketides produced by fungal
species such as:
Aspergillus flavus
A. parasiticus
rarely A. nomius (Payne and Brown, 1998).
 Under humid conditions, these fungi are produced in
Groundnuts, corn/ maize, sorghum, rice, spices and other agricultural products
Livestock feeds
Medicinal herbs
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7. Aflatoxins?...
 Aflatoxin contamination
can occur at any stage of food production
from pre-harvest to storage stages of the food chain
 Aflatoxin accumulation is dependent on
Environmental factors such as:
 moisture
 temperature
 plant density
 poor harvest practices and
 Improper grain storage
3

8. Aflatoxins?...
 The removal of aflatoxins is very difficult due to their
stability and thermal resistance in dried products
melting points > 250oC
stable at a pH range of 3 to 10.
resistant to food processing
 Thus remain unchanged throughout the food chain.
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9. Classification of Aflatoxins
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 There are more than 20 known aflatoxins, but the four main ones are
aflatoxin B1 (AFT-B1)
aflatoxin B2 (AFT-B2)
aflatoxin G1 (AFT-G1)
aflatoxin G2 (AFT-G2)
 Named based on their
 blue (B) or green (G) fluorescence under UV light
 relative mobility, 1 and 2 (higher and lower respectively), by TLC
AFTs B series are produced by
 A. flavus, A. parasiticus and A. nomius
AFTs G series are produced by
 A. parasiticus and A. nomius

10. Classification of Aflatoxins…
 Other members of the aflatoxin family
 AFT-M1 and AFT-M2
are oxidative forms of AFT-B1
modified in the digestive tract of animals
isolated from milk, urine and feces
 The level of toxicity associated with aflatoxin varies with the types present
 AFT-B1 > AFT-G1 > AFT-B2 > AFT-G2
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11. Health Consequences of Aflatoxins
 Aflatoxins are of great concern to public health because of their ability to
accumulate in the body
found in edible tissues of animals, such as liver and muscles
found in animal food products such as milk and eggs
found in human maternal breast milk and maternal cord blood
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12. Health Consequences…
 Exposure to aflatoxin can lead to several health-related conditions including
 acute and chronic aflatoxicosis
 aflatoxin-related immune suppression
 liver disease (liver cancer and liver cirrhosis)
 adverse pregnancy outcomes, including
 intrauterine growth restriction
 premature delivery
 pregnancy loss
 nutrition-related problems in children such as stunted growth
 mutagenesis
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13. ORIGIN OF AFLATOXINS
• Historical Background of Aflatoxins
• Potential Sources of Aflatoxins
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14. Historical Background of Aflatoxins
Late 1950s and early 1960s
• Turkey “X” disease in the UK, more than 100, 000 died
•Resulted from A. flavus highly infested peanut feed
•Understanding towards AFTs came to light
•Name was given by designating ‘‘A’’ for the genus Aspergillus and ‘‘FLA’’ for the species flavus and toxins
In 1974
• Outbreak of hepatitis due to aflatoxin in the states of Gujrat and Rajasthan, and North west
part of India
• About 106 deaths
• Aflatoxin contained in the staple food prepared by maize
In 1981
• Major Aflatoxin outbreak occurred in East Province of Kenya
• Occurred because of consumption of home grown maize contaminated with
molds
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15. Historical Background…
In 1963
Structural characterization and synthesis of the major AFTs
In 1968-1985
Evaluation of the association of AFTs ingestion and HCC incidence in humans
Since 1985
Different organizations and countries set regulatory strategies
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16. Potential Sources of Aflatoxins
 The major sources of aflatoxins are fungi such as
Aspergillus flavus
Aspergillus parasiticus
Aspergillus nomius
 Taxonomically, these fungal species are from:
 Phylum: Ascomycota
 Class: Eurotiomycetes
 Order: Eurotiales
 Family: Trichocomaceae
(French Agency for Food, Environmental and Occupational Health & Safety, 2012)10
the most commonly found in several foods

17. Potential Sources…
 The fungal species infect many crops in the field, during harvest, in storage,
and during processing.
A. flavus is dominant in corn, cottonseed, and tree nuts
A. parasiticus is dominant in peanuts.
A. nomius has been reported from tree nuts, sugarcane and insects such
as alkali bees
 They can grow at temperatures ranging between 12 and 48oC and 40
degrees latitude north and south
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18. BIOSYNTHESIS OF AFLATOXINS
• Chemistry of Aflatoxins
• Biosynthetic Pathway of Aflatoxins
• Factors affecting Aflatoxin Synthesis
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19. Chemistry of Aflatoxins
 AFTs are difuranocoumarin derivatives
in which a bifuran group is attached at one side of the coumarin nucleus.
 AFTs B series
 pentanone ring is attached to the other side
 AFTs G series
 a six-membered lactone ring is attached(Kumar et al. 2016)
 AFTs M1 and M2 (milk toxins)
 bear a hydroxy group at the junction of the two furan rings (Bräse, 2013)
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20. Chemistry…
Structures were revealed by Büchi
 In 1963 (AFT-B1 and G1)
 In 1965 (AFT-B2 and AFT-G2 )
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21. Chemistry…
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Physicochemical properties of aflatoxins
 Form colourless to pale-yellow crystals
 Intensely fluorescent in ultraviolet light
 Slightly soluble in water (10–30μg/mL)
 Insoluble in non-polar solvents
 Freely soluble in moderately polar organic solvents
 Examples: methanol and especially in dimethyl sulfoxide (IARC, 2002).

22. Biosynthesis of Aflatoxins
 The biosynthesis of AFTs takes place by a polyketide metabolic pathway
Consists at least 18 multienzymatic conversion reactions
As many as 30 genes are also potentially involved
A positive regulatory gene, aflR,
required for transcriptional activation of most of the aflatoxin structural
genes
 Biosynthesis starts from a hexanoyl-CoA starter and seven malonyl-CoA
extender units
 Formation of a poly-β-keto chain
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23. Biosynthesis…
23
acetyl-CoA + 2 ×
malonyl-CoA
NADPH
versicolorin B

24. Biosynthesis…
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25. Factors Affecting Aflatoxin Biosynthesis
 The major factors that influence aflatoxin biosynthesis
 Nutritional factors
 Including carbon and nitrogen sources
 Environmental factors
 Including water activity and temperature
 Developmental factors
 Including sporulation and sclerotial formation
 Plant metabolites
 Including n-decyl aldehyde, Octanal, hexanal, 13(S)-hydroperoxide derivative of linoleic
acid15

26. Factors …
I. Nutritional factors
 Simple sugars such as glucose, sucrose, fructose, and maltose
support aflatoxin formation
while peptone, sorbose, and lactose do not
 The sources of nitrogen such as
Asparagine, aspartate, alanine, ammonium nitrate, ammonium nitrite,
ammonium sulfate, glutamate, glutamine, and proline containing media
 support aflatoxin production
while sodium nitrate and sodium nitrite containing media do not
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27. Factors…
II. Environmental factors
 The fungi are capable of growing over a wide pH range (Yu, 2012).
 AFTs synthesis optimally occurs in the pH range of 3.4–5.5.
 The optimum temperature for AFTs production is between 24° and 30°C
 The higher the water activity, the better are the conditions for fungal
growth and toxin synthesis (Caira et al. 2012).
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28. Factors…
III. Developmental stage
 Spore formation and secondary metabolite formation occur at about the same
time.
 Some mutants that are deficient in sporulation
 Are unable to produce aflatoxins
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29. Factors…
IV. Plant metabolites
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Metabolites Fungal growth AFTs production
n-decyl aldehyde Reduces Reduces by 95%
Octanal Reduces by 60% Increases by 500%
Hexanal Reduces by 50% No effect
13(S)-hydroperoxide
derivative of linoleic
acid
Reduces Reduces

30. ASSOCIATION OF AFLATOXINS WITH CANCER
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31. Association of Aflatoxins with Cancer
 The IARC (1993) classified AFTs as group I carcinogens
 The most potent naturally occurring human hepatocarcinogens
 AFT-B1 is the most potent
 Direct correlation b/n AFT degree of exposure and incidence of HCC
(with odds ratio 3.74:1.0 to 10.86:1.0) (Kew, 2013)
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HCC
• Accounts for about 9.2% of all new cancers worldwide
• Approximately 84% of all new cases occur in resource limited regions
• Ranked third in annual cancer mortality rates
• About 93% of patients dying within 12 months of the onset of symptoms

32. Association …
 AFTs poisoning in the presence of HBV infection
Increased risk of HCC development
30X greater than the risk in individuals exposed to aflatoxin only
AFTs with chronic HBV act as cofactor for HCC development
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33. Association…
Mechanism of AFT induced carcinogenesis
 Hepatic enzymes metabolize AFTs, generating reactive epoxide species
 The epoxide species form a covalent bond with guanine
 Results in DNA adducts
 The adducts produce mutation in the p53 tumor-suppressor gene
 a G-to-T transversion at codon 249
 biomarker for aflatoxin contamination
 unscheduled DNA synthesis and chromosomal strand breaks in human cells
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34. REGULATIONS OF AFLATOXINS
• Regulations of aflatoxins
• Determination of aflatoxins
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35. Regulations of Aflatoxins
 When AFTs are confirmed to cause cancer, countries established various
regulations (>75 countries) (Ali et al. 2005).
Either total AFTs or AFT-B1
 In view of toxic and carcinogenic effects of aflatoxin contaminated foods,
USDA and FDA set the tolerance limits for AFB1 and total AFTs in food
 AFT-B1 - 5μg/kg
 Total AFTs - 20μg/kg
The Codex Alimentarius Commission, adopted a limit of
15μg/kg for total AFTs
10μg/kg total AFTs in ready-to-eat almonds, hazelnuts, and pistachios (Siddique et
al. 2013).
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36. Regulations…
 EU countries allow much lower permitted concentration of aflatoxins
AFT-B1 - 2μg/kg
Total AFTs - 4μg/kg
 AFTs remains largely unregulated throughout Africa.
 As of 2003, aflatoxin regulations existed for five countries (Van
Egmond and Jonker, 2004)
 Ethiopia has no aflatoxin and other mycotoxin regulation (Wolde, 2016).
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37. Regulations…
Country Food items Limits(μg/kg)
USA Nuts, figs, and related products 2-12
UK Milk, dairy products, barley, cocoa, coffee, corn,
peanuts, oats, rice, sorghum, soy, tree nuts, and
wheat
0.5-20
EU Groundnut, peanuts, spices and other food stuff 2-12
Australia All the food stuff except peanuts 5
Peanuts and related products 15
India Nuts, spices, cereals, and all other food products 30
China Rice, sorghum, barley, nuts etc. 5-50
Japan All of the food stuffs 10
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Permissible limits of aflatoxins in different countries (Sarma et al. 2017)

38. Determination of Aflatoxins
 The determination of AFTs has been carried out using
 TLC
 HPTLC
 HPLC
 LC–MS
 immunological methods
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Designed to detect the possible presence of
AFTs B1, B2, G1 and G2 (Wolde, 2017).

39. Determination…
 The WHO quality control for herbal materials guideline (1998)
 Recommended to use HPLC system based tests
 Why HPLC is recommended?
 It does not require the use of toxic solvents
 such as chloroform and dichloromethane
 It uses a multifunctional column, which contains lipophilic and charged
active sites
 It uses fluorescence detection to determine aflatoxins B1, B2, G1 and G2
 It has high precision, high sensitivity, and high automation
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40. Future directions
 A holistic and multidisciplinary approach is required to mitigate the exposure
to this toxin
 Increase awareness of AFTs and support risk mitigation practices
 Future epidemiologic studies of HCC should focus on good practices
in food preparation
food storage
consumption of aflatoxin containing foods and milk
 In Ethiopia
more emphasis to develop regulatory actions and preliminary prevention
strategies40

41. Conclusion
 AFTs are the most well-known and researched mycotoxins.
 Maize and groundnut are the most contaminated commodities
 Like the food items medicinal herbs can also be infected by these pathogens
 Contamination is more common in developing countries than the developed
countries
 AFT-B1 is the most potent AFT to cause HCC and other adverse health
effects to human and animal.
 Even though it is common in Ethiopia, the country has no aflatoxin and
other mycotoxins regulatory systems
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42. References
 Aflatoxins review (final).pdf
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